Characterization of the TRBP domain required for Dicer interaction and function in RNA interference
Tóm tắt
Dicer, Ago2 and TRBP are the minimum components of the human RNA-induced silencing complex (RISC). While Dicer and Ago2 are RNases, TRBP is the double-stranded RNA binding protein (dsRBP) that loads small interfering RNA into the RISC. TRBP binds directly to Dicer through its C-terminal domain. We show that the TRBP binding site in Dicer is a 165 amino acid (aa) region located between the ATPase and the helicase domains. The binding site in TRBP is a 69 aa domain, called C4, located at the C-terminal end of TRBP. The TRBP1 and TRBP2 isoforms, but not TRBPs lacking the C4 site (TRBPsΔC4), co-immunoprecipitated with Dicer. The C4 domain is therefore necessary to bind Dicer, irrespective of the presence of RNA. Immunofluorescence shows that while full-length TRBPs colocalize with Dicer, TRBPsΔC4 do not. tarbp2-/- cells, which do not express TRBP, do not support RNA interference (RNAi) mediated by short hairpin or micro RNAs against EGFP. Both TRBPs, but not TRBPsΔC4, were able to rescue RNAi function. In human cells with low RNAi activity, addition of TRBP1 or 2, but not TRBPsΔC4, rescued RNAi function. The mapping of the interaction sites between TRBP and Dicer show unique domains that are required for their binding. Since TRBPsΔC4 do not interact or colocalize with Dicer, we suggest that TRBP and Dicer, both dsRBPs, do not interact through bound dsRNA. TRBPs, but not TRBPsΔC4, rescue RNAi activity in RNAi-compromised cells, indicating that the binding of Dicer to TRBP is critical for RNAi function.
Tài liệu tham khảo
Hammond SM: Dicing and slicing: the core machinery of the RNA interference pathway. FEBS Lett. 2005, 579 (26): 5822-5829.
Liu Q, Rand TA, Kalidas S, Du F, Kim HE, Smith DP, Wang X: R2D2, a bridge between the initiation and effector steps of the Drosophila RNAi pathway. Science. 2003, 301 (5641): 1921-1925.
Forstemann K, Tomari Y, Du T, Vagin VV, Denli AM, Bratu DP, Klattenhoff C, Theurkauf WE, Zamore PD: Normal microRNA maturation and germ-line stem cell maintenance requires Loquacious, a double-stranded RNA-binding domain protein. PLoS Biol. 2005, 3 (7): e236-
Bernstein E, Caudy AA, Hammond SM, Hannon GJ: Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature. 2001, 409 (6818): 363-366.
Jaskiewicz L, Filipowicz W: Role of Dicer in posttranscriptional RNA silencing. Curr Top Microbiol Immunol. 2008, 320: 77-97.
Zamore PD, Haley B: Ribo-gnome: the big world of small RNAs. Science. 2005, 309 (5740): 1519-1524.
Tahbaz N, Kolb FA, Zhang H, Jaronczyk K, Filipowicz W, Hobman TC: Characterization of the interactions between mammalian PAZ PIWI domain proteins and Dicer. EMBO Rep. 2004, 5 (2): 189-194.
Liu J, Carmell MA, Rivas FV, Marsden CG, Thomson JM, Song JJ, Hammond SM, Joshua-Tor L, Hannon GJ: Argonaute2 is the catalytic engine of mammalian RNAi. Science. 2004, 305 (5689): 1437-1441.
Bernstein E, Kim SY, Carmell MA, Murchison EP, Alcorn H, Li MZ, Mills AA, Elledge SJ, Anderson KV, Hannon GJ: Dicer is essential for mouse development. Nat Genet. 2003, 35 (3): 215-217.
Chendrimada TP, Gregory RI, Kumaraswamy E, Norman J, Cooch N, Nishikura K, Shiekhattar R: TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature. 2005, 436 (7051): 740-744.
Haase AD, Jaskiewicz L, Zhang H, Lainé S, Sack R, Gatignol A, Filipowicz W: TRBP, a regulator of cellular PKR and HIV-1 virus expression, interacts with Dicer and functions in RNA silencing. EMBO Rep. 2005, 6 (10): 961-967.
Rossi JJ: Mammalian Dicer finds a partner. EMBO Rep. 2005, 6 (10): 927-929.
Castanotto D, Sakurai K, Lingeman R, Li H, Shively L, Aagaard L, Soifer H, Gatignol A, Riggs A, Rossi JJ: Combinatorial delivery of small interfering RNAs reduces RNAi efficacy by selective incorporation into RISC. Nucleic Acids Res. 2007, 35 (15): 5154-64.
Parker GS, Maity TS, Bass BL: dsRNA Binding Properties of RDE-4 and TRBP Reflect Their Distinct Roles in RNAi. J Mol Biol. 2008, 384 (4): 967-979.
MacRae IJ, Ma E, Zhou M, Robinson CV, Doudna JA: In vitro reconstitution of the human RISC-loading complex. Proc Natl Acad Sci USA. 2008, 105 (2): 512-517.
Bannwarth S, Gatignol A: HIV-1 TAR RNA: the target of molecular interactions between the virus and its host. Curr HIV Res. 2005, 3: 61-71.
Daher A, Longuet M, Dorin D, Bois F, Segeral E, Bannwarth S, Battisti PL, Purcell DF, Benarous R, Vaquero C, Meurs EF, Gatignol A: Two dimerization domains in the trans-activation response RNA-binding protein (TRBP) individually reverse the protein kinase R inhibition of HIV-1 long terminal repeat expression. J Biol Chem. 2001, 276 (36): 33899-33905.
Duarte M, Graham K, Daher A, Battisti P-L, Bannwarth S, Segeral E, Jeang K-T, Gatignol A: Characterization of TRBP1 and TRBP2: stable stem-loop structure at the 5' end of TRBP2 mRNA resembles HIV-1 TAR and is not found in its processed pseudogene. J Biomed Sci. 2000, 7: 494-506.
Gatignol A, Buckler-White A, Berkhout B, Jeang KT: Characterization of a human TAR RNA-binding protein that activates the HIV-1 LTR. Science. 1991, 251 (5001): 1597-1600.
Gatignol A, Duarte M, Daviet L, Chang YN, Jeang KT: Sequential steps in Tat trans-activation of HIV-1 mediated through cellular DNA, RNA, and protein binding factors. Gene Expr. 1996, 5 (4–5): 217-228.
Chang KY, Ramos A: The double-stranded RNA-binding motif, a versatile macromolecular docking platform. FEBS J. 2005, 272 (9): 2109-2117.
Daviet L, Erard M, Dorin D, Duarte M, Vaquero C, Gatignol A: Analysis of a binding difference between the two dsRNA-binding domains in TRBP reveals the modular function of a KR-helix motif. Eur J Biochem. 2000, 267 (8): 2419-2431.
Erard M, Barker DG, Amalric F, Jeang KT, Gatignol A: An Arg/Lys-rich core peptide mimics TRBP binding to the HIV-1 TAR RNA upper-stem/loop. J Mol Biol. 1998, 279 (5): 1085-1099.
Gatignol A, Buckler C, Jeang KT: Relatedness of an RNA-binding motif in human immunodeficiency virus type 1 TAR RNA-binding protein TRBP to human P1/dsI kinase and Drosophila staufen. Mol Cell Biol. 1993, 13 (4): 2193-2202.
Laraki G, Clerzius G, Daher A, Melendez-Pena C, Daniels S, Gatignol A: Interactions between the double-stranded RNA-binding proteins TRBP and PACT define the Medipal domain that mediates protein-protein interactions. RNA Biol. 2008, 5 (2): 92-103.
Daher A, Laraki G, Singh M, Melendez-Pena CE, Bannwarth S, Peters AH, Meurs EF, Braun RE, Patel RC, Gatignol A: TRBP control of PACT-induced phosphorylation of PKR is reversed by stress. Mol Cell Biol. 2009, 29 (1): 254-265.
Lee JY, Kim H, Ryu CH, Kim JY, Choi BH, Lim Y, Huh PW, Kim YH, Lee KH, Jun TY, Rha HK, Kang JK, Choi CR: Merlin, a tumor suppressor, interacts with transactivation-responsive RNA-binding protein and inhibits its oncogenic activity. J Biol Chem. 2004, 279 (29): 30265-30273.
Bannwarth S, Lainé S, Daher A, Grandvaux N, Clerzius G, LeBlanc AC, Hiscott J, Gatignol A: Cell-specific regulation of TRBP1 promoter by NF-Y transcription factor in lymphocytes and astrocytes. J Mol Biol. 2006, 355 (5): 898-910.
Bannwarth S, Talakoub L, Letourneur F, Duarte M, Purcell DF, Hiscott J, Gatignol A: Organization of the human tarbp2 gene reveals two promoters that are repressed in an astrocytic cell line. J Biol Chem. 2001, 276 (52): 48803-48813.
Christensen HS, Daher A, Soye KJ, Frankel LB, Alexander MR, Laine S, Bannwarth S, Ong CL, Chung SW, Campbell SM, Purcell DF, Gatignol A: Small interfering RNAs against the TAR RNA binding protein, TRBP, a Dicer cofactor, inhibit human immunodeficiency virus type 1 long terminal repeat expression and viral production. J Virol. 2007, 81 (10): 5121-5131.
Ong CL, Thorpe JC, Gorry PR, Bannwarth S, Jaworowski A, Howard JL, Chung S, Campbell S, Christensen HS, Clerzius G, Mouland AJ, Gatignol A, Purcell DF: Low TRBP levels support an innate human immunodeficiency virus type 1 resistance in astrocytes by enhancing the PKR antiviral response. J Virol. 2005, 79 (20): 12763-12772.
Zhong J, Peters AH, Lee K, Braun RE: A double-stranded RNA binding protein required for activation of repressed messages in mammalian germ cells. Nat Genet. 1999, 22 (2): 171-174.
Boden D, Pusch O, Silbermann R, Lee F, Tucker L, Ramratnam B: Enhanced gene silencing of HIV-1 specific siRNA using microRNA designed hairpins. Nucleic Acids Res. 2004, 32 (3): 1154-8.
Perron MP, Provost P: Protein interactions and complexes in human microRNA biogenesis and function. Front Biosci. 2008, 13: 2537-2547.
Provost P, Dishart D, Doucet J, Frendewey D, Samuelsson B, Radmark O: Ribonuclease activity and RNA binding of recombinant human Dicer. EMBO J. 2002, 21 (21): 5864-5874.
Zhang H, Kolb FA, Brondani V, Billy E, Filipowicz W: Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP. EMBO J. 2002, 21 (21): 5875-5885.
Cummins JM, He Y, Leary RJ, Pagliarini R, Diaz LA, Sjoblom T, Barad O, Bentwich Z, Szafranska AE, Labourier E, Raymond CK, Roberts BS: et al, The colorectal microRNAome. Proc Natl Acad Sci USA. 2006, 103 (10): 3687-3692.
Battisti PL, Daher A, Bannwarth S, Voortman J, Peden KW, Hiscott J, Mouland AJ, Benarous R, Gatignol A: Additive activity between the trans-activation response RNA-binding protein, TRBP2, and cyclin T1 on HIV type 1 expression and viral production in murine cells. AIDS Res Hum Retroviruses. 2003, 19 (9): 767-78.
Castanotto D, Li H, Rossi JJ: Functional siRNA expression from transfected PCR products. RNA. 2002, 8 (11): 1454-1460.
Good PD, Krikos AJ, Li SX, Bertrand E, Lee NS, Giver L, Ellington A, Zaia JA, Rossi JJ, Engelke DR: Expression of small, therapeutic RNAs in human cell nuclei. Gene Ther. 1997, 4 (1): 45-54.
Sambrook J, Fritsch EF, Maniatis T: Molecular cloning: a laboratory manual. 1989, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press,